Metabolically stabilized double stranded mRNA

This application is a division of U.S. patent application Ser. No. 16/090,468, filed on Oct. 1, 2018, which application is a U.S. National Stage Filing under 35 U.S.C. 371 from International Application No. PCT/US2017/025527, filed on Mar. 31, 2017, and published as WD 2017/173354 on Oct. 5, 2017, which application claims the benefit of the filing date of U.S. application Ser. No. 62/317,142, filed on Apr. 1, 2016, and U.S. application Ser. No. 62/335,186, filed on May 12, 2016, the disclosures of which are incorporated by reference herein.

This invention was made with government support under contract GM097093, GM117785 and GM008365 awarded by the National Institutes of Health. The government has certain rights in the invention.

The development of a non-viral gene delivery system that efficiently expresses proteins in the liver has been a long-sought goal for over twenty-five years (Wu et al., 1988). Preclinical studies have demonstrated that protein expression in hepatocytes could lead to curative treatments for liver metabolic diseases as well as diseases in other organs (Wooddell et al., 2013; Chuah et al., 2013; Richard et al., 2009). Much of the effort in developing a non-viral gene delivery system for the liver has focused on packaging and targeting plasmid DNA (Pun et al., 2002; Lenter et al., 2004; Read et al., 2005). Despite much effort, systemic delivery of DNA formulations resulted in either negligible or very low gene transfer efficiency in liver hepatocytes (Hu et al., 2013). In contrast, hydrodynamic delivery of naked plasmid DNA to liver achieves expression efficiency equivalent to adenovirus or adeno-associated virus (AAV) (Liu et al., 1999). While hydrodynamic delivery is highly efficient because it overcomes the rate limiting step of delivery of DNA to the nucleus, it is also an invasive delivery method requiring both high volume and pressure (Al Dosari et al., 2005; Zhang et al., 2004; Andrianaivo et al., 2004; Hodges et al., 2003). Alternatively, the delivery of mRNA to the cytosol leading to translation, circumvents the need for delivery to the nucleus. Despite this major advantage, the rapid metabolism of mRNA by ubiquitous RNase remains a significant hurdle to achieving efficient expression of systemically delivered mRNA gene delivery systems (Sahin et al., 2014).

Since the earliest report demonstrating in vivo expression following intramuscularly (i.m.) dosed naked mRNA (Wolff et al., 1990), numerous studies have attempted to increase the stability and expression efficiency of mRNA formulations using cationic lipids (Deering et al., 2014; Phua et al., 2013; Schlake et al., 2012; Kariko et al, 2012; Malone et al., 1989). Intratracheal high pressure spraying of an mRNA Megafectin™ lipoplex resulted in transfection of the lung (Kormann et al., 2011), whereas regeneration following myocardial infarction was achieved by intracardial injection of RNAiMAX™ mRNA (Zangi et al., 2013). Stemfect™ mRNA delivered nasally resulted in tumor vaccination (Phua et al., 2014). Alternatively, systemically delivered Stemfect™ mRNA produced low level expression in the spleen (Phua et al., 2013). While these studies demonstrate that mRNA lipoplexes possess improved in vivo gene transfer over naked mRNA, their efficiency in vivo is still very low due to relatively weak ionic binding of cationic lipids to mRNA. A mannosylated histidinylated lipoplex dosed systemically resulted in expression in spleen macrophages which primed a tumor vaccine response (Perche et al., 2011).

In an attempt to further improve mRNA stability, nanoparticle delivery systems have been developed and tested in vitro (Avci-Adali et al., 2014; Cheng et al., 2012; Debus et al., 2010) and in vivo (Perche et al., 2011; Wang et al., 2013; Uchida et al., 2013). Systemic delivery of targeted stealth mRNA lipoplexes in vivo led to transfection efficiency similar to DNA formulations in solid tumor (Wang et al., 2013). Intrathecally dosed mRNA polyplex nanomicelles produced measurable expression in the cerebrospinal fluid (Uchida et al., 2013). Notably, none of the mRNA cationic lipid or nanoparticle formulations reported to date were able to transfect liver.

There have been only two reports of successful liver transfection with mRNA (McCaffrey et al., 2002; Wilber et al., 2006). The expression of mRNA in the liver was first achieved by McCaffrey et al. (2002) who measured luciferase expression by bioluminescence imaging (BLI) in mice following hydrodynamic (HD)-dosing of 50 μg of naked mRNA to detect low level expression (10photons/sec/cm/steradian). The transient expression in the liver was only detectable at 3 hours and required the co-administration of 30 μg of decoy RNA and 400 units of RNase inhibitor. In an attempt to improve transfection efficiency, Wilber et al. (2006) refined the mRNA by inserting 5′ and 3′□-globin untranslated regions (UTRs) flanking luciferase to increase mRNA cellular half-life (Malone et al., 1989). HD-dosing of 50 □g of UTR mRNA resulted in a 15-fold increase in the expression efficiency at 3 hours relative to mRNA lacking UTRs (Wilber et al., 2006) but failed to significantly extend the expression. Co-administration of decoy mRNA and RNase inhibitors significantly improved efficiency but failed to extend peak expression past 12 hours. While these reports demonstrate the feasibility of expressing proteins in the liver when HD-dosing mRNA, the efficiencies reported are far below that achievable with plasmid DNA due to mRNA's susceptibility to metabolism during delivery.

As shown herein, double stranded (ds) mRNA is much more metabolically stable than single-stranded (ss) mRNA and so ds mRNA formulations as described herein, can be dosed intravenously and circulate in the blood. ds mRNA is also as efficiently translated into protein as single-stranded mRNA. Thus, ds mRNA that includes single-stranded mRNA may be employed in targeted gene delivery system, e.g., systemic delivery, to express therapeutic proteins in animals, e.g., humans. Persistent expression is achieved by self-amplifying mRNA constructs designed to replicate mRNA in the cytosol and extend its expression.

In particular, as described below, the expression efficiency in liver following hydrodynamic delivery of in vitro transcribed ds mRNA was improved using an exemplary codon-optimized mRNA luciferase construct with flanking 3′ and 5′ human β-globin untranslated regions (UTR mRNA) over an un-optimized mRNA without β-globin UTRs.

In one embodiment, the disclosure provides isolated double stranded (ds) mRNA encoding a protein of interest, which ds mRNA is highly stable to degradation, e.g., after treatment with RNase or incubation in serum. At least one strand of the ds mRNA has a 5′ cap, a start codon, and a polyA sequence, and this strand encodes a protein. The two strands of the ds mRNA are hydrogen bonded (Watson Crick) over at least 10 nucleotides and up to the full length of the shortest strand, if the strands are of different lengths. For example, the two strands of the ds mRNA are hydrogen bonded over at least 25, 50, 100, 200, 500, 1000, 2000 or more, e.g., 10,000 nucleotides (or any integer between 25 and 10,000), or over at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or more of the length of at least one strand. In one embodiment, at least one strand may include one or more non-natural nucleotides, e.g., a nucleotide that has a non-natural sugar, a non-natural nucleotide base, a non-phosphodiester bond between nucleotides, or any combination thereof. In one embodiment, at least one of the strands may be formed using one or more of 2′-fluoro-2′deoxycytidine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-methylcytidine-5;-triphosphate, 2′-O-methylcytidine-5′-triphosphate, 2′-amino-2′-deoxycytidine-5′-triphosphate, 2′-amino-2′-deoxycytidine-5′-triphosphate, 2′-azido-2′-deoxycytidine-5′-triphosphate, aracytidine-5′-triphosphate, 2-thiocytidine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 3′-O-methylcytidine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, pseudoisocytidine-5′-triphosphate, N-methylcytidine-5′-triphosphate, 5-carboxycytidine-5′-triphosphate, 5-formylcytidine-5′-triphosphate, 5-hydroxymethylcytidine-5′-triphosphate, 5-hydroxycytidine-5′-triphosphate, 5-methoxycytidine-5′-triphosphate, thienocytidine-5′-triphosphate, cytidine-5′-triphosphate, 3′-deoxycytidine-5′-triphosphate, biotin-16-aminoallylcytidine-5′-triphosphate, cyanine 3-aminoallylcytidine-5′-triphosphate, cyanine 5-aminoallylcytidine-5′-triphosphate or cytidine-5′-O-(1-thiotriphosphate). In one embodiment, at least one of the strands is formed using one or more of 2′-fluoro-2′-deoxyuridine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 2′-O-methyluridine-5′-triphosphate, pseudouridine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 2′-amino-2′-deoxyuridine-5′-triphosphate, 2′-azido-2′-5′-triphosphate, 2-thiouridine-5′-triphosphate, arauridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, 6-azauridine-5′-triphosphate, 2′-O-methylpseudouridine-5′-triphosphate, 2′-O-methyl-5-methyluridine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 3′-O-methyluridine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, N-methylpseudouridine-5′-triphosphate, 5,6-dihydro-5-methyluridine-5′-triphosphate, 5-hydroxymethyluridine-5′-triphosphate, 5-formyluridine-5′-triphosphate, 5-carboxyuridine-5′-triphosphate, 5-hydroxyuridine-5′-triphosphate, 5-methoxyuridine-5′-triphosphate, thienouridine-5′-triphosphate, 5-carboxymethylesteruridine-5′-triphosphate, uridine-5′-triphosphate, 3′-deoxy-5-methyluridine-5′-triphosphate, 3′-deoxyuridine-5′-triphosphate, biotin-16-aminoallyluridine-5′-triphosphate, desthiobiotin-16-aminoallyl-uridine-5′-triphosphate, cyanine 3-aminoallyluridine-5′-triphosphate, cyanine 7-aminoallyluridine-5′-triphosphate or uridine-5′-O-(1-thiotriphosphate). In one embodiment, at least one of the strands is formed using one or more of 5-aminoallyl-CTP, 2-amino-ATP, 5-Br-UTP, 5-carboxy-CTP, 5-carboxy-UTP, 5-carboxymethyest-UTP, 7-deaza-ATP, 5-formyl-CTP, 5-formyl-UTP, 5-hydroxy-CTP, 5-hydroxy-UTP, 5-hydroxymethyl-CTP, 5-hydroxymethyl-UTP, 5-iodo-UTP, 5-methoxy-CTP, 5-methoxy-UTP, N6-methyl-amino-ATP, N6-methyl-ATP, 5-methyl-CTP, pseudo-UTP, thieno-CTP, thieno-GTP, 1-thio-ATP or 2-thio-UTP. In one embodiment, one of the strands includes 5-formyl cytidine or pseudouridine. In one embodiment, at least 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90% or more of the nucleotides are non-natural nucleotides, and in one embodiment, the strands are hydrogen bonded over at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of the strands.

Further provided is a method to prevent, inhibit or treat a disorder in a mammal associated with an absence or deficiency in a protein or in a mammal in need of increased amounts of a protein. The method includes systemically administering to the mammal an effective amount of a composition comprising one or more distinct ds mRNA as described above. In one embodiment, the composition is employed to express human factor VIII (HFVIII) in liver hepatocytes for treating hemophilia A. In one embodiment, the composition may be employed to systemically deliver CRISPR Cas9 or other gene editing systems.

Also provided are methods of making a ds mRNA encoding a protein of interest. In one embodiment, a strand of mRNA having a 5′ cap, a start codon, a polyA sequence and an open reading frame for the protein and a strand of RNA that has sequence complementarity with the mRNA over at least 10 nucleotides are provided. The mRNA and the RNA with sequence complementarity are allowed to hydrogen bond, thereby providing the ds mRNA. In one embodiment, the strands are provided by transcription of one or more vectors, e.g. a plasmid vector. In one embodiment, the strands are provided by transcription of a single vector that includes an open reading frame for the protein that is flanked by a first promoter positioned to express the strand of mRNA and a second promoter positioned to express the strand of RNA with sequence complementarity. In one embodiment, at least one of the strands includes one or more non-natural nucleotides or nucleotide modifications. In one embodiment, the one or more nucleotide modifications are introduced post-synthesis of at least one of the strands. In one embodiment, the one or more non-natural nucleotides are incorporated during synthesis of at least one of the strands. In one embodiment, the strands are hydrogen bonded over at least 90% of the length of the strands. In one embodiment, the strands are hydrogen bonded over the entire length of the strands. In one embodiment, wherein the strands are not the same length. For example, when hybridized, the 3′ end of the RNA with sequence complementarity overhands the 5′ end of the strand of mRNA, or the 3′ end of the RNA with sequence complementarity is recessed relative to the 5′ end of the strand of mRNA. In one embodiment, the strands are the same length. In one embodiment, at least one of the strands is synthesized in an in vitro transcription reaction. In one embodiment, at least one of the strands is synthesized in a cell.

Further provided is a method of using the ds mRNA, e.g., to express a protein of interest. In one embodiment, a composition comprising a ds mRNA encoding the protein of interest, wherein at least one strand of the ds mRNA has a 5′ cap, a start codon, a polyA sequence and encodes the protein, wherein the two strands of the ds mRNA are hydrogen bonded over at least 10 nucleotides is provided and the composition is introduced to cells in an amount effective to express the protein. In one embodiment, the cells are in a mammal for example, the composition is systemically administered to the mammal. In one embodiment, the composition is locally administered to the mammal. In one embodiment, the protein is a therapeutic protein. In one embodiment, the protein is for cancer immunotherapy. In one embodiment, the protein is a cancer antigen. In one embodiment, the protein is a nuclease. In one embodiment, the protein is a microbial protein, for instance, one useful for immunization. In one embodiment, the composition further comprises a carrier protein. In one embodiment, the composition further comprises a synthetic polymer optionally in combination with a carrier protein. In one embodiment, the composition further comprises a liposome. In one embodiment, the ds mRNA forms a nanoparticle, e.g., optionally in combination with a carrier protein, lipid, such as a lipid bilayer surrounding the ds mRNA, or synthetic polymer. In one embodiment, the nanoparticle has a diameter of about 1 nm to about 500 nm, about 50 nm to about 250 nm, or about 100 nm to about 200 nm. In one embodiment, the ds mRNA forms a microparticle, e.g., optionally in combination with a carrier protein, lipid, such as a lipid bilayer surrounding the ds mRNA, or synthetic polymer. In one embodiment, the microparticle has a diameter of about 0.5 μm to about 500 μm, about 10 μm to about 30 μm, or about 20 μm to about 40 μm.

Various non-viral vectors can be used to deliver DNA, mRNA and short double-stranded RNA, including small interfering RNA (siRNA) and microRNA (miRNA) mimics. However, delivery of double stranded RNA (not mRNA, siRNA or miRNA) is highly toxic to cells due to triggering of apoptosis. Moreover, in order to be useful for gene therapy, the vectors need to avoid degradation by serum endonucleases and evade immune detection. They also need to avoid renal clearance from the blood and prevent nonspecific interactions.

A stabilized ds mRNA containing composition is disclosed herein that is useful for prophylactic or therapeutic gene delivery. The compositions may be employed in methods to prevent, inhibit or treat a disorder or disease in a mammal, such as a canine, feline, bovine, porcine, equine, caprine, ovine, or human, which disorder or disease is amenable to treatment with one or more exogenously delivered genes. For example, the disorder or disease may be associated with a decreased amount of a gene product, the absence of a gene product, or the presence of an aberrant gene product, e.g., one having no activity, aberrant activity, reduced activity or increased activity relative to a mammal without the disorder or disease.

Exemplary Disorders or Diseases for Use with the Compositions

The compositions may be employed to prevent, inhibit or treat a variety of disorders or diseases associated with a deficiency in (or absence of) a protein or an aberrant protein (e.g., with low or no activity or excessive or unregulated activity) (see Table 1 for a list of monogenic disorders). Genes that may be employed include but are not limited to those that prevent, inhibit or treat hemophilia, anemia or other blood disorders, cancer, cardiovascular disease, lysosomal storage diseases, musculoskeletal diseases, neurodegenerative diseases, respiratory disease, and the like. Exemplary genes are shown in Table 2.

Hemophilia-F8, F9, F11, VWF

Hemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation, which is used to stop bleeding when a blood vessel is broken. Like most recessive sex-linked. X chromosome disorders, hemophilia is more likely to occur in males than females. For example, Hemophilia A (clotting factor VIII deficiency), the most common form of the disorder, is present in about 1 in 5,000-10,000 male births. Hemophilia 8 (factor IX deficiency) occurs in around 1 in about 20,000-34,000 male births. Hemophilia lowers blood plasma clotting factor levels of the coagulation factors, e.g. F8, needed for a normal clotting process. Thus when a blood vessel is injured, a temporary scab does form, but the missing coagulation factors prevent fibrin formation, which is necessary to maintain the blood clot F8, for example, encodes Factor VIII (FVIII), an essential blood clotting protein. Factor VIII participates in blood coagulation, it is a cofactor for factor IXa which, in the presence of Caand phospholipids forms a complex that converts factor X to the activated form Xa.

Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating F8 for the treatment and/or prevention of diseases associated with reduced F8 expression or function such as hemophilia. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating F9 for the treatment and/or prevention of diseases associated with reduced F9 expression or function such as hemophilia. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating F11 for the treatment and/or prevention of diseases associated with reduced F11 expression or function such as hemophilia. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating VWF for the treatment and/or prevention of diseases associated with reduced VFW expression or function such as Von Willebrand's Disease

Thus, in one embodiment, the compositions may be employed to prevent, inhibit or treat hemophilia including but not limited to hemophilia A, characterized by low levels of or the absence of factor 8 (Also called FVIII or factor VIII deficiency), hemophilia B, characterized by low levels of or the absence of factor 9 (Also called FIX or factor IX deficiency), hemophilia C, characterized by low levels of or the absence of factor 11 (Also called FXI or factor XI deficiency), or Von Willebrands Disease, characterized by a deficiency of a blood clotting protein Von Willebrand factor.

Lysosomal Storage Diseases

In one embodiment, the compositions may be employed to prevent, inhibit or treat a lysosomal storage disease. Lysosomal storage diseases include, but are not limited to, mucopolysaccharidosis (MPS) diseases, for instance, mucopolysaccharidosis type I, e.g., Hurler syndrome and the variants Scheie syndrome and Hurler-Scheie syndrome (a deficiency in alpha-L-iduronidase); Hunter syndrome (a deficiency of iduronate-2-sulfatase); mucopolysaccharidosis type III, e.g., Sanfilippo syndrome (A, B, C or D; a deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV e.g., mucopolysaccharidosis type IV, e.g., Morquio syndrome (a deficiency of galactosamine-6-sulfate sulfatase or beta-galactosidase); mucopolysaccharidosis type VI, e.g., Maroteaux-Lamy syndrome (a deficiency of arylsulfatase B); mucopolysaccharidosis type II; mucopolysaccharidosis type III (A, B, C or D; a deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV (A or B; a deficiency of galactosamine-6-sulfatase and beta-galatacosidase); mucopolysaccharidosis type VI (a deficiency of arylsulfatase B); mucopolysaccharidosis type VII (a deficiency in beta-glucuronidase); mucopolysaccharidosis type VIII (a deficiency of glucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IX (a deficiency of hyaluronidase); Tay-Sachs disease (a deficiency in alpha subunit of beta-hexosaminidase); Sandhoff disease (a deficiency in both alpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis (type I or type II); Fabry disease (a deficiency in alpha galactosidase); metachromatic leukodystrophy (a deficiency of aryl sulfatase A); Pompe disease (a deficiency of acid maltase); fucosidosis (a deficiency of fucosidase); alpha-mannosidosis (a deficiency of alpha-mannosidase); beta-mannosidosis (a deficiency of beta-mannosidase), ceroid lipofuscinosis, and Gaucher disease (types I, II and III; a deficiency in glucocerebrosidase), as well as disorders such as Hermansky-Pudlak syndrome; Amaurotic idiocy; Tangier disease; aspartylglucosaminuria; congenital disorder of glycosylation, type Ia; Chediak-Higashi syndrome; macular dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickel syndrome; Farber lipogranulomatosis; fibromatosis; geleophysic dysplasia; glycogen storage disease I; glycogen storage disease Ib; glycogen storage disease Ic; glycogen storage disease III; glycogen storage disease IV; glycogen storage disease V; glycogen storage disease VI; glycogen storage disease VII; glycogen storage disease 0; immunoosseous dysplasia, Schimke type; lipidosis; lipase b; mucolipidosis II, including the variant form; mucolipidosis IV; neuraminidase deficiency with beta-galactosidase deficiency; mucolipidosis I; Niemann-Pick disease (a deficiency of sphingomyelinase); Niemann-Pick disease without sphingomyelinase deficiency (a deficiency of a npc1 gene encoding a cholesterol metabolizing enzyme); Refsum disease; Sea-blue histiocyte disease; Infantile sialic acid storage disorder; sialuria; multiple sulfatase deficiency; triglyceride storage disease with impaired long-chain fatty acid oxidation; Winchester disease; Wolman disease (a deficiency of cholesterol ester hydrolase); Deoxyribonuclease I-like 1 disorder; arylsulfatase E disorder; ATPase, H+ transporting, lysosomal, subunit 1 disorder; glycogen storage disease lib; Ras-associated protein rab9 disorder; chondrodysplasia punctata 1, X-linked recessive disorder; glycogen storage disease VIII; lysosome-associated membrane protein 2 disorder; Menkes syndrome; congenital disorder of glycosylation, type Ic; and sialuria.

Cancer-SERPINF1, BCL2L11, BRCA1, RB1, ST7

In one embodiment, the compositions may be employed to prevent, inhibit or treat cancer. Cancer is a broad group of various diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. Several genes, many classified as tumor suppressors, are down-regulated during cancer progression. e.g., SERPINF1, BCL2L11, BRCA1, RB1, and ST7, and have roles in inhibiting genomic instability, metabolic processes, immune response, cell growth/cell cycle progression, migration, and/or survival. These cellular processes are important for blocking tumor progression. SERPINF1 encodes an anti-angiogenic factor BCL2L11 encodes an apoptosis facilitator. BRCA1 encodes a RING finger protein involved in DNA damage repair. RB1 prevents excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. ST7 suppresses tumor growth in mouse models and is involved in regulation of genes involved in differentiation. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating SERPINF1, BCL2L11, BRCA1, RB1, and ST7 for the treatment and/or prevention of diseases associated with reduced SERPINF1, BCL2L11, BRCA1, RB1, and ST7 expression or function such as cancer. For example, aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating BCL2L11 for the treatment or prevention of human T-cell acute lymphoblastic leukemia and lymphoma. In another example, aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating BRCA1 for the treatment or prevention of breast cancer or pancreatic cancer. In another example, aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating RB1 for the treatment or prevention of bladder cancer, osteosarcoma, retinoblastoma, or small cell lung cancer. In another example, aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating ST7 for the treatment or prevention of myeloid cancer, head and neck squamous cell carcinomas, breast cancer, colon carcinoma, or prostate cancer.

Examples of cancer include but are not limited to leukemias, lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervous system cancers and genito-urinary cancers, in some embodiments, the cancer is adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenomas. Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer. Ewing family tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumors. Kaposi sarcoma, kidney cancer, renal cell cancer, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, melanoma. Merkel cell carcinoma, malignant mesothelioma, squamous neck cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous neck cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Wilms tumor.

Fragile X Syndrome—FMR1

Fragile X syndrome (FXS) (also known as Martin-Bell syndrome, or Escalante's syndrome) is a genetic syndrome that is the most common known single-gene cause of autism and the most common inherited cause of intellectual disability. It results in a spectrum of intellectual disability ranging from mild to severe as well as physical characteristics such as an elongated face, large or protruding ears, and larger testes (macroorchidism), behavioral characteristics such as stereotypical movements (e g hand-flapping), and social anxiety. Fragile X syndrome is associated with the expansion of the CGG trinucleotide repeat affecting the Fragile X mental retardation 1 (FMR1) gene on the X chromosome, resulting reduced expression of the X mental retardation protein (FMRP), which is required for normal neural development. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating FMR1 for the treatment and/or prevention of diseases associated with reduced FMR1 expression or function such as Fragile X syndrome.

Premature Ovarian Failure—FMR1

Premature Ovarian Failure (POF), also known as premature ovarian insufficiency, primary ovarian insufficiency, premature menopause, or hypergonadotropic hypogonadism, is the loss of function of the ovaries before age 40. POF can be associated mutations in the Fragile X mental retardation 1 (FMR1) gene on the X chromosome, resulting reduced expression of the X mental retardation protein (FMRP). Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating FMR1 for the treatment and/or prevention of diseases associated with reduced FMR1 expression or function such as Premature Ovarian Failure.

Obesity-FNDC5, GCK, ADIPOQ

Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems. A person is considered obese when his or her weight is 20% or more above normal weight. The most common measure of obesity is the body mass index or BMI. A person is considered overweight if his or her BMI is between 25 and 29.9; a person is considered obese if his or her BMI is over 30 Obesity increases the likelihood of various diseases, particularly heart disease, type 2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis. Obesity is most commonly caused by a combination of excessive food energy intake, lack of physical activity, and genetic susceptibility. Overexpression of FNDC5, fibronectin type II containing 5, has been shown in animal models to reduce body weight in obese mice. GCK, glucokinase (hexokinase 4), phosphorylates glucose to produce glucose-6-phosphate, the first step in most glucose metabolism pathways. Mutations in the GCK gene have been found to be associated with obesity in humans. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating FNDC5 for the treatment and/or prevention of diseases associated with reduced FNDC5 expression or function such as obesity. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GCK for the treatment and/or prevention of diseases associated with reduced GCK expression or function such as obesity.

Adiponectin, encoded by the ADIPOQ gene, is a hormone that regulates metabolism of lipids and glucose. Adipocytes found in adipose tissue secrete adiponectin into the bloodstream where it self-associates into larger structures by binding of multiple adiponectin trimers to form hexamers and dodecamers. Adiponectin levels are inversely related to the amount of body fat in an individual and positively associated with insulin sensitivity both in healthy subjects and in diabetic patients. Adiponectin has a variety of protective properties against obesity-linked complications, such as hypertension, metabolic dysfunction, type 2 diabetes, atherosclerosis, and ischemic heart disease through its anti-inflammatory and anti-atherogenic properties. Specifically with regard to type 2 diabetes, administration of adiponectin has been accompanied by a reduction in plasma glucose and an increase in insulin sensitivity. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating ADIPOQ for the treatment and/or prevention of diseases associated with reduced ADIPOQ expression or function such as obesity or an obesity-linked disease or disorders such as hypertension, metabolic dysfunction, type 2 diabetes, atherosclerosis, and ischemic heart disease.

Type 2 Diabetes—FNDC5, GCK, GLP1R, SIRT1, ADIPOQ

Type 2 diabetes (also called Diabetes mellitus type 2 and formally known as adult-onset diabetes) a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Type 2 diabetes makes up about 90% of cases of diabetes with the other 10% due primarily to diabetes mellitus type 1 and gestational diabetes. Obesity is thought to be the primary cause of type 2 diabetes in people who are genetically predisposed to the disease. The prevalence of diabetes has increased dramatically in the last 50 years. As of 2010 there were approximately 285 million people with the disease compared to around 30 million in 1985. Overexpression of FNDC5, fibronectin type II containing 5, has been shown in animal models to improve their insulin sensitivity GCK, glucokinase (hexokinase 4), phosphorylates glucose to produce glucose-6-phosphate, the first step in most glucose metabolism pathways. Mutations in the GCK gene are known to be associated with Type 2 Diabetes. Glucagon-like peptide 1 receptor (GLP1R) is known to be expressed in pancreatic beta cells. Activated GLP1R stimulates the adenylyl cyclase pathway which results in increased insulin synthesis and release of insulin SIRT1 (Sirtuin 1, also known as NAD-dependent deacetylase sirtuin-1) is an enzyme that deacetylates proteins that contribute to cellular regulation. Sirtuin 1 is downregulated in cells that have high insulin resistance and inducing its expression increases insulin sensitivity, suggesting the molecule is associated with improving insulin sensitivity. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating FNDC5 for the treatment and/or prevention of diseases associated with reduced FNDC5 expression or function such as Type 2 Diabetes. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GCK for the treatment and/or prevention of diseases associated with reduced GCK expression or function such as Type 2 Diabetes. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GLP1R for the treatment and/or prevention of diseases associated with reduced GLP1R expression or function such as Type 2 Diabetes. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating SIRT1 for the treatment and/or prevention of diseases associated with reduced SIRT1 expression or function such as Type 2 Diabetes. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating ADIPOQ for the treatment and/or prevention of diseases associated with reduced ADIPOQ expression or function such as Type 2 Diabetes.

Metabolic Disease—IGF1, SIRT1

Inborn errors of metabolism comprise a large class of genetic diseases involving disorders of metabolism. The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances (substrates) into others (products). In most of the disorders, problems arise due to accumulation of substances which are toxic or interfere with normal function, or to the effects of reduced ability to synthesize essential compounds. Inborn errors of metabolism are now often referred to as congenital metabolic diseases or inherited metabolic diseases. IGF-1, Insulin growth factor-1, is a hormone similar in molecular structure to insulin. IGF-1 plays an important role in childhood growth and continues to have anabolic effects in adults. Reduced IGF-1 and mutations in the IGF-1 gene are associated with metabolic disease SIRT1 (Sirtuin 1, also known as NAD-dependent deacetylase sirtuin-1) is an enzyme that deacetylates proteins that contribute to cellular regulation. SIRT1 has been shown to de-acetylate and affect the activity of both members of the PGC1-alpha/ERR-alpha complex, which are essential metabolic regulatory transcription factors. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IGF-1 for the treatment and/or prevention of diseases associated with reduced IGF-1 expression or function such as metabolic disease. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating SIRT1 for the treatment and/or prevention of diseases associated with reduced SIRT1 expression or function such as metabolic disease.

Aging/Senescence—SIRT1

Senescence is the state or process of aging. Cellular senescence is a phenomenon where isolated cells demonstrate a limited ability to divide in culture, while organismal senescence is the aging of organisms. After a period of near perfect renewal (in humans, between 20 and 35 years of age), organismal senescence/aging is characterised by the declining ability to respond to stress, increasing homeostatic imbalance and increased risk of disease. This currently irreversible series of changes inevitably ends in death. SIRT1 (Sirtuin 1, also known as NAD-dependent deacetylase sirtuin-1) is an enzyme that deacetylates proteins that contribute to cellular regulation. Mice overexpressing SIRT1 present lower levels of DNA damage, decreased expression of the ageing-associated gene p16Ink4a, a better general health and fewer spontaneous carcinomas and sarcomas. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating SIRT1 for the treatment and/or prevention of biological processes associated with reduced SIRT1 expression or function such as aging.

Autoimmune—GRN, IDO1, CD274

Autoimmune diseases arise from an inappropriate immune response of the body against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. Autoimmune diseases are classified by corresponding types of hypersensitivity: type II, type III, or type IV. Examples of autoimmune disease include, but are not limited to, Ankylosing Spondylitis. Autoimmune cardiomyopathy. Autoimmune hemolytic anemia, Autoimmune hepatitis. Autoimmune inner ear disease, immune lymphoproliferative syndrome, Autoimmune peripheral neuropathy. Autoimmune pancreatitis. Autoimmune polyendocrine syndrome, Autoimmune thrombocytopenic purpura. Celiac disease, Cold agglutinin disease, Contact dermatitis, Crohn's disease, Dermatomyositis, Diabetes mellitus type 1, Eosinophilic fasciitis, Gastrointestinal pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Idiopathic thrombocytopenic purpura, Lupus erythematosus, Miller-Fisher syndrome, Myasthenia gravis, Pemphigus vulgaris, Pernicious anaemia, Polymyositis, Primary biliary cirrhosis, Psoriasis, Psoriatic arthritis, Relapsing polychondritis, Rheumatoid arthritis, Sjögren's syndrome, Temporal arteritis, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease, Vasculitis, Vitiligo, and Wegener's granulomatosis, IDO1 encodes indoleamine 2,3-dioxygenase (IDO)—a heme enzyme that catalyzes the first and rate-limiting step in tryptophan catabolism to N-formyl-kynurenine. This enzyme acts on multiple tryptophan substrates including D-tryptophan. L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin. This enzyme is thought to play a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity, increased catabolism of tryptophan by IDO1 suppresses T cell responses in a variety of diseases or states, including autoimmune disorders. GRN encodes a precursor protein called Progranulin, which is then cleaved to form the secreted protein granulin. Granulin regulates cell division, survival, motility and migration. Granulin has roles in cancer, inflammation, host defense, cartilage development and degeneration, and neurological functions. Downregulation of GRN has been shown to increase the onset of autoimmune diseases like rheumatoid arthritis. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IDO1 for the treatment and/or prevention of diseases associated with reduced IDO1 expression or function such as autoimmune diseases. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GRN for the treatment and/or prevention of diseases associated with reduced GRN expression or function such as autoimmune diseases.

CD274 (also known as PDL1) is a transmembrane protein containing IgV-like and IgC-like extracellular domains expressed on immune cells and non-hematopoietic cells, and is a ligand for the programmed death receptor (PD-1) expressed on lymphocytes and macrophages. PD-1 and CD274 interactions are essential in maintaining the balance of T-cell activation, tolerance, and immune-mediated tissue damage. CD274 is involved in inhibiting the initial phase of activation and expansion of self-reactive T cells, and restricting self-reactive T-cell effector function and target organ injury. More specifically, activation of PD-1 by CD274 inhibits T-cell proliferation, cytokine production, and cytolytic function by blocking the induction of phosphatidylinositol-3-kinase (PI3K) activity and downstream activation of Akt.

Decreased expression of CD274 results in autoimmunity in animal models. For example, mice deficient for the CD274 receptor, PD-1, developed features of late onset lupus. In another instance, blockade of CD274 activity in a mouse model of Type 1 diabetes resulted in accelerated progression of diabetes. In yet another example, CD274 blockade in an animal model of multiple sclerosis resulted in accelerated disease onset and progression.

Increasing expression of CD274 offers a novel approach for treating diseases related to inappropriate or undesirable activation of the immune system, including in the context of translation rejection, allergies, asthma and autoimmune disorders. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating CD274 for the treatment and/or prevention of diseases associated with reduced CD274 expression or function such as autoimmune disease, transplant rejection, allergies or asthma.

Inflammation (Chronic Inflammation)—GRN, IDO1, IL10

Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. However, chronic inflammation can also lead to a host of diseases, such as hay fever, periodontitis, atherosclerosis, and rheumatoid arthritis. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Inflammatory disorder include, but are not limited to: acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplantation rejection (graft vs host disease), vasculitis and interstitial cystitis.

GRN encodes a precursor protein called Progranulin, which is then cleaved to form the secreted protein granulin. Granulin regulates cell division, survival, motility and migration. Granulin has roles in cancer, inflammation, host defense, cartilage development and degeneration, and neurological functions. GRN has been shown to alleviate inflammatory arthritis symptoms in mouse models. Indoleamine 2,3-dioxygenase 1 (IDO1; previously referred as IDO or INDO) is the main inducible and rate-limiting enzyme for the catabolism of the amino acid tryptophan through the kynurenine pathway. Increased catabolism of tryptophan by IDO1 suppresses T cell responses in a variety of diseases, such as allograft rejection.

Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GRN for the treatment and/or prevention of diseases associated with reduced GRN expression or function such as chronic inflammation. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GRN for the treatment and/or prevention of diseases associated with reduced GRN expression or function such as rheumatoid arthritis. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IDO1 for the treatment and/or prevention of diseases associated with reduced IDO1 expression or function such as chronic inflammation. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IDO1 for the treatment and/or prevention of diseases associated with reduced IDO1 expression or function such as graft vs host disease.

IL-10 is capable of inhibiting synthesis of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-3, TNFα and GM-CSF made by cells such as macrophages and regulatory T-cells. It also displays a potent ability to suppress the antigen-presentation capacity of antigen presenting cells. Treatment with IL-10 (e.g. as a recombinant protein given to patients) is currently in clinical trials for Crohn's disease. Genetic variation in the IL-10 pathway modulates severity of acute graft-versus-host disease. Mouse models of arthritis have been shown to have decreased levels of IL-10. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating GRN for the treatment and/or prevention of diseases associated with reduced GRN expression or function such as chronic inflammation.

Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IL-10 for the treatment and/or prevention of diseases associated with reduced IL-10 expression or function such as chronic inflammation. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IL-10 for the treatment and/or prevention of diseases associated with reduced IL-10 expression or function such as rheumatoid arthritis. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IL-10 for the treatment and/or prevention of diseases associated with reduced IL-10 expression or function such as graft vs host disease. Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating IL-10 for the treatment and/or prevention of diseases associated with reduced IL-10 expression or function such as Crohn's disease.